Impact of Inhibitor Size and Flexibility on the Binding Pathways to c-Src Kinase

Abstract The conformational plasticity of protein kinases poses a challenge for inhibitor design, motivating the use of molecular dynamics (MD) simulations to study their dynamic binding processes. While conformational changes are increasingly discussed, the impact of drug compound flexibility remains underexplored because of experimental limitations. In this study, we employ two-dimensional replica-exchange MD simulations to investigate how c-Src kinase binds to PP1, a small inhibitor, and to dasatinib, a larger, more flexible inhibitor. Simulations totaling 600 μs revealed frequent binding and unbinding events, yielding statistically converged information about the binding pathways. While both inhibitors follow multiple binding pathways, a notable difference emerges in their binding mechanisms on the free-energy profiles: PP1 is rigid along the binding path, whereas dasatinib substantially changes its conformation at different stages in the pathway to the canonical pose. Conformational analysis reveals distinct conformers of dasatinib, including a hidden intermediate, which helps to avoid trapping at the salt-bridge pair linking the β3 strand and the αC-helix of the c-Src kinase. These results, distinct from previous computational studies, demonstrate that inhibitor size and flexibility affect binding mechanisms and have implications on kinetics.